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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
one-generation reproductive toxicity
Remarks:
based on test guideline (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
April-August 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well conducted study according to GLP
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report Date:
2011

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 415 [One-Generation Reproduction Toxicity Study (before 9 October 2017)]
Deviations:
yes
Remarks:
see below
Principles of method if other than guideline:
This study was carried out as an extended OECD 422 study in which 12 animals per sex per group were exposed 10 weeks (instead of 2 weeks) prior to mating so that male fertility could be examined. In doing so the study became more a one-generation test (OECD 415) than a combined subacute/reproscreening test (OECD 422). However, 12 animals/sex/group were used (at least 10 animals/sex/group) to comply to the REACH requirement for Annex VII and VIII studies.
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Chemical name: Sodium hydrogen [N,N-bis[2-[bis(carboxymethyl)amino]ethyl]glycinato(5-)]ferrate(2-)
Purity: 11.6% (Fe content)
Batch no: CFC 9883
Expiry date: 1 January 2013

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutshland, Sulzfeld, Germany
- Age at study initiation: 5 weeks (females), 6 weeks (males)
- Weight at study initiation: mean weight males ca. 170 g; mean weight females ca. 106 g
- Fasting period before study: not applicable
- Housing: 4 per sex in macrolon cages, with wood shavings as bedding material, and paper strips as environmental enrichment
- Use of restrainers for preventing ingestion (if dermal): not applicable
- Diet (e.g. ad libitum): ad lib
- Water (e.g. ad libitum): ad lib
- Acclimation period: one week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±2 degrees C, reaching a minimum of 19.2 degrees C
- Humidity (%): at least 45% and not exceeding 65%. During several periods, humidity was outside the limits reaching a minimum of 43% and a maximum of 96% during a short period
- Air changes (per hr): ca. 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 7 April To: 4 August 2010

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Weekly, one bottle of test formulation per dose level was prepared. Preparation of the test formulations was performed one day before the first day of the dosing period and at weekly intervals thereafter until completion of the dosing phase of the study. The different concentrations of the test substance in tap water were prepared by stirring on a magnetic stirrer for at least 1h. The pH of the test formulations of groups 2, 3 and 4 were set between pH 6-7 using sodium carbonate (Na2CO3). Subsequently, under continuous stirring, 8 aliquots (7 days plus 1 extra) were taken according to the volume required for each dosing. Aliqouts were stored in a refrigerator in the dark. On each subsequent day, one aliquot for each group was removed from the refrigerator and allowed to equilibrate to ambient temperature. All aliquots were continuously stirred on a magnetic stirrer during the entire administration period in order to maintain the homogeneity of the test substance in the vehicle.
Sodium carbonate was added to all three test formulations to adjust the acidity of the formulations to pH 6-7: it appeared that the amount of test substance used for the preparation of the test solution for the high-dose group did not dissolve unless the pH was adjusted. On the first 2 days of the study, animals of the low- and mid-dose groups were treated with test formulations without the addition of sodium carbonate. From Day 2 onwards, all animals of the low-, mid- and high-dose groups were treated with test formulations with added sodium carbonate. In week 2 of the study, the pH of the test formulations of the low-, mid- and high-dose groups were marginally higher (resp 7.03, 7.05 and 7.06). This also applied for week 8 of the study, regarding test formulations of the low- and mid-dose groups (resp 7.18 and 7.01).

VEHICLE: tap water
- Concentration in vehicle: 0, 15, 50 and 150 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg bw
Details on mating procedure:
- M/F ratio per cage: 1
- Length of cohabitation: max 16 days. At the end of the pre-mating period (23 June 2010), each female was caged with one male from the same group. Since the number of females that was mated after 1 week was relatively low in all groups, the mating period was extended initially with another week. Since on Day 12 of the mating period, the number of females that was mated was still relatively low in all groups, males that had not mated during these 12 days were replaced by males of the same dosing group that already had mated with another female. The newly formed pairs were allowed to mate up to another 4 days (1 oestrus cycle).
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility: not done.
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged: individually
- Any other deviations from standard protocol: no
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
To determine the homogeneity and content of DTPA-FeNaH in gavage liquid, iron was used as a marker for the test item. Iron concentrations in gavage liquid were determined using inductively coupled plasma atomic emission spectroscopy (ICP-AES).
The concentrations of iron measured in the gavage liquids prepared on 15 April 2010, 15 June 2010 and 06 July 2010 were ‘close to intended’ (relative difference < 10 %) for all gavage liquids at all dose levels, except for the low-dose level gavage liquids prepared on 15 April 2010 (+10.7%) and the low-, mid- and high-dose level gavage liquids prepared on 06 July 2010 (+10.1%, +11.0% and +13.6%, respectively).
Duration of treatment / exposure:
10 weeks pre-mating, 16 days mating, 3 weeks gestation, and 4 days lactation
Frequency of treatment:
single daily application by gavage (parental animals)
Details on study schedule:
- F1 parental animals not mated until [...] weeks after selected from the F1 litters: not applicable as F1 animals were killed on postnatal day 4.
- Selection of parents from F1 generation when pups were [...] days of age: not applicable as F1 animals were killed on postnatal day 4.
- Age at mating of the mated animals in the study: 15-16 weeks
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 150, 500 and 1500 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
12
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on studies done with EDTA and EDTA-MnA2
- Rationale for animal assignment (if not random): computer randomization proportionately to BW

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: observations outside the home cage were made once weekly; FOB and motor activity were assessed in week 8 of the pre-mating period.

BODY WEIGHT: Yes
- Time schedule for examinations: weekly (males and females) and on day 1 and 4 of lactation (females)

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined: weekly (at same time as measurement of bw)

WATER CONSUMPTION: No

Oestrous cyclicity (parental animals):
Not measured
Sperm parameters (parental animals):
Parameters examined:
testis weight, epididymis weight: 12 rats/group
sperm count in epididymides, sperm motility, sperm morphology: 5 rats/group
sperm count in testes, daily sperm production: 5 rats/group
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no, because this screening study was ended on day 4 post-partum

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities,and skeletal analysis

GROSS EXAMINATION OF DEAD PUPS:
yes, for external abnormalities

Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals as soon as possible after mating (at least 13 weeks of treatment)
- Maternal animals: All surviving animals at or shortly after day 4 of lactation (almost 14 weeks of treatment)

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera

ORGAN WEIGHTS:
- testes, epididymides (12 rats/group)
- adrenals, brain, heart, kidneys, liver, spleen, thymus (5 rats/sex/group)

HISTOPATHOLOGY:
- ovaries, uterus (12 rats/group; control and high dose group
- testes, epididymides, seminal vesicles, prostate, coagulating glands (12 rats/group; control and high dose group
- adrenals, axillary lymph nodes, brain, caecum, colon, femur, Peyer's patches, heart, kidneys, liver, lungs, mesenteric lymph nodes, peripheral nerve, rectum, small intestines, spinal cord, spleen, stomach, thymus, thyroid, trachea/bronchi, urinary bladder (5 rats/sex/group; control and high dose group)

Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring was sacrificed at 4 days of age.
- These animals were subjected to postmortem examinations (macroscopic) externally for gross abnormalities

GROSS NECROPSY
- Gross necropsy consisted of external examinations; pups were stored in a freezer for possible skeletal analyses (not done).

ORGAN WEIGHTS: not done

HISTOPATHOLOGY: not done

SKELETAL ANALYSIS: done
Statistics:
- Clinical findings were evaluated by Fisher's exact probability test.
- Body weight, body weight gain, food consumption and organ weights data were subjected
to one-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparison tests.
- Fisher's exact probability test was used to evaluate the number of mated and pregnant
females, the number of pregnant females with implants but no pups, females with live pups,
females with stillborn pups, live and dead fetuses or pups and the numbers of litters lost
entirely.
- Pre-coital time (mean number of days), the duration of gestation, the number of corpora
lutea and implantation sites, the total number of pups delivered (mean), the mean number
of live pups per litter and pre- and post-implantation loss (%) were evaluated by Kruskal-
Wallis nonparametric analysis of variance and by the Mann-Whitney U test.
- Mortality data and data of the pathology of parent animals were evaluated by the Fisher’s
exact probability test.
- Sperm parameters were evaluated by one-way analysis of variance followed by Dunnett’s
multiple comparison test (epididymal and testicular sperm count and numerical sperm
motility parameters) or by Kruskal-Wallis non parametric analysis of variance and by
Mann-Whitney U test (motility parameters expressed as a percentage and sperm
morphology).
Reproductive indices:
- pre-coital time = time between the start of mating and successful copulation
- duration of gestation = time between gestation day 0 and day of delivery
- mating index= (number of females mated/number of females placed with males) x 100
- male fertility index = (number of males that became sire/number of males placed with females) x 100
- female fertility index = (number of pregnant females/number of females placed with males) x 100
- female fecundity index = (number of pregnant females/number of females mated) x 100
- gestation index = (number of females with live pups or pups/number of females pregnant) x 100
- pre-implantation loss = [(number of corpora lutea – number of implantation sites)/number of corpora lutea] x 100
- number of lost implantations = number of implantations sites - number of pups born alive
- post-implantation loss = [(number of implantation sites - number of pups born alive)/number of implantation sites] x 100
Offspring viability indices:
- live birth index = (number of pups born alive/number of pups born) x 100
- viability index day n-m= (number of pup surviving m days/number of liveborn on day n) x100
- pup mortality day n = (number of dead pups on day n/total number of pups on day n) x 100
- sex ratio day n = (number of live male fetuses or pups on day n/ number of live fetuses or pups on day n) x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
effects observed, treatment-related
Reproductive performance:
no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY: Soft faeces were observed in males and females of the high-dose groups in various weeks of the pre-mating period . This finding was most pronounced in the first week of the study. Soft feaces were also observed in some rats of the mid-dose group in the first week of the study only. The findings in the high-dose group occasionally reached the level of statistical significance. The repeated occurrence of soft faeces in the high-dose group was considered treatment-related.
One female of the low-dose group (no. 33) was found dead on day 64 of the pre-mating period. In the fourth week of the study, one female of the mid-dose group (no. 57) showed a hunched body position, piloerection and was thin and weak. On Day 32 of the pre-mating period it was found dead. On Day 3 of the gestation period, a female of the high dose group (no. 85) was killed because it was thin and showed piloerection and abnormal respiration (rales, dyspnoea, decreased frequency). The death of females nos 33 and 85 is likely to have been caused by misdosing. The cause of death of female no. 57 of the mid-dose group could not be examined at necropsy because its organs were already autolytic.

BODY WEIGHT AND FOOD CONSUMPTION: The mean body weight of males of the high-dose group was decreased when compared to the control group, reaching the level of statistical significance as from Day 35 of the study. In addition, the mean body weight change of males of the high-dose group was statistically significantly decreased at various time periods (Days 0-7, 28-49, 63-70). These observed effects on body weight were considered to be related to treatment. Food consumption was not affected by treatment.

TEST SUBSTANCE INTAKE: no effects (gavage)

ESTROUS CYCLE: not examined

SPERM MEASURES: The percentage of motile epididymal sperm cells was statistically significantly decreased in males of the high-dose group. This finding was accompanied by a decrease in the percentage of progressive epididymal sperm cells and an increase in the percentage of static cells. Although the latter two findings did not reach the level of statistical significance, the observed effects were ascribed to treatment. The weight of the cauda epididymides of males of the high-dose group was statistically significantly decreased. In addition, the sperm reserve (sperm count per (3) ml) of males of the high-dose group was statistically significantly decreased. These effects were considered to be related to treatment. No differences were observed on sperm morphology between the control group and the high dose group. No statistical significant differences were found on testicular parenchyma weight, on the number of spermatozoa or on the daily sperm production.

ORGAN WEIGHTS: The relative weights of the kidneys and liver of male and female animals of the high-dose group were statistically significantly increased. These findings were considered treatmentrelated. The absolute weight of the epididymides was decreased in males of the mid- and high-dose
group. The relative weight of this organ was decreased in males of the high-dose group but not in males of the mid-dose group. Therefore, only the decreased weight of the epididymides of males of the high-dose group was considered to be related to treatment.

GROSS PATHOLOGY: no effects

HISTOPATHOLOGY (NON-NEOPLASTIC): no treatment-related effects

HISTOPATHOLOGY (NEOPLASTIC): no changes

HISTORICAL CONTROL DATA: not needed

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: decreased relative weight of epididymides, a decrease in sperm motility and epididymal sperm reserve

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed

Details on results (F1)

VIABILITY (OFFSPRING): no effects

CLINICAL SIGNS (OFFSPRING): no effects

BODY WEIGHT (OFFSPRING): no effects

SEXUAL MATURATION (OFFSPRING): not done, pups were necropsied on day 4 post partum

ORGAN WEIGHTS (OFFSPRING): not done

GROSS PATHOLOGY (OFFSPRING): Stillborn pups of the mid- and high-dose groups and one pup of the high-dose group that died during the lactation period were examined macroscopically. None of the pups showed any remarkable abnormality.

HISTOPATHOLOGY (OFFSPRING): no effects

OTHER FINDINGS (OFFSPRING): skeletal examinations. No skeletal malformations or anomalies were found in any of the pups of any groups. Analysis of skeletal variations and retardations did not reveal treatment-related effects.

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
>= 1 500 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no treatment-related effects

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Based on the results of this study, viz. a decrease in sperm motility, cauda epididymis weight and in sperm reserve, as observed in male animals treated with the highest concentration of the test substance, the No Observed Adverse Effect Level (NOAEL) for fertility is 500 mg/kg body weight/day.
Executive summary:

The objective of this study was to provide data on the possible effects of the test substance DTPA-FeNaH on reproductive performance of Wistar rats and the development of pups following daily oral administration at concentrations of 0, 150, 500 or 1500 mg/kg bw of the test substance by gavage to male and female rats during a pre-mating period of 10 weeks, during mating (16 days), and during gestation and lactation until postnatal Day 4 (PN Day 4); see also section 7.5.1 and 7.8.2.

The homogeneity and content of the test substance in the gavage solutions were confirmed by analysis.

Males and females of the high-dose group showed soft faeces in various weeks of the premating period. Daily clinical observations during the gestation and lactation period did not reveal any treatment-related changes in the animal’s appearance, general condition or behaviour. Mean body weights were decreased in males of the high-dose group from week 5 onwards. There were no treatment-related effects on female body weights during the entire study. No treatment-related effects were observed on food consumption of male and female animals during the entire study.

No treatment-related effects were observed on pre-coital time, mating index, female fecundity index, male and female fertility indices, duration of gestation, number of animals that delivered liveborn pups, number of corpora lutea, number of implantation sites, pre-implantation loss or viability index. Decreased epididymal sperm motility, decreased weight of the cauda epididymides and decreased epididymal sperm reserve were observed in males of the high-dose group. No changes were observed in epididymal sperm morphology, or the weight of testicular parenchyma, the number of spermatozoa per gram testicular parenchyma or on the daily sperm production. The relative weights of the kidneys and liver were increased in both sexes of the high-dose group. The relative weight of the epididymides was decreased in males of the high-dose group. Macroscopic examination at necropsy, microscopic examination of organs and tissues did not reveal any treatment-related changes

Based on the results of this study, viz. soft faeces (both sexes), decreased body weight gain (males), prolonged prothrombin time (males), increased haemoglobin concentration (males), decreased ALAT activity and chloride concentration (males) and increased relative weights of kidneys and liver (both sexes) as observed in animals treated with the highest concentration of the test substance, the No Observed Adverse Effect Level (NOAEL) for parental toxicity is 500 mg/kg body weight/day. Based on the results of this study, viz. a decrease in sperm motility, cauda epididymis weight and in sperm reserve, as observed in male animals treated with the highest concentration of the test substance, the No Observed Adverse Effect Level (NOAEL) for fertility is 500 mg/kg body weight/day. Based on the results of this study, which did not show any toxicological effects of the test substance on development, the No Observed Adverse Effect Level (NOAEL) for developmental toxicity is ≥1500 mg/kg body weight/day.